Mass spectrometry is a long established technique for identification and quantitation of often complex mixtures of large organic molecules. In recent years, techniques have been developed that allow analysis of a wide range of both biological and non-biological materials, with application across the fields of law enforcement (e.g. identification of drugs and explosives materials), environmental, scientific research, and biology (e.g. in proteomics, the study of simple and complex mixtures of proteins, with applications in drug discovery, disease identification and so forth).
Proteins, comprising large numbers of amino acids, are typically of significant molecular weight. Thus accurate identification and quantitation of the protein by direct mass spectrometric measurement is challenging. It is thus well known to carry out fragmentation of the precursor sample material. A variety of fragmentation techniques is known, which may result in the generation of different fragment ions from the precursor ions. Moreover, the fragmentation mechanism may be affected by different applied fragmentation energies.
A number of different arrangements are known for the production of fragment ions from precursors. For example, the TSQ™ triple quadruple group of devices sold by Thermo Fisher Scientific LLC comprises three quadrupoles supplied with sample ions usually from a liquid chromatograph (LC), for tandem mass spectrometry. The first quadrupole acts as a mass filter to select a range of precursor ions supplied from the LC. The second quadrupole causes fragmentation of the ions selected by the first quadrupole, typically through the use of a collision gas. The third quadrupole then acts as a mass filter, typically in scan mode, to allow a mass spectrum of abundance versus m/q (mass-to-charge ratio) to be obtained.
Instead of a triple quadrupole system, the final stage of mass analysis after fragmentation may be carried out using other devices, such as a Time-of-Flight mass spectrometer, a Fourier transform Ion Cyclotron Resonance (FT-ICR) Mass spectrometer, an orbital trapping mass spectrometer such the Orbitrap® or the like. Each different final stage mass analyser has its own advantages and disadvantages with respect, for example, to mass resolution, duty cycle, sensitivity, cost and so forth.
Analysis of samples can broadly be separated into data independent analysis/acquisition (DIA) and data dependent analysis/acquisition (DDA). DIA seeks to determine what is present in a sample of potentially unknown identity. To determine the molecular structure of sample molecules, the first mass filter in a tandem mass spectrometer is set to pass all ions within a selected range of m/z. This range of precursor ions is then fragmented in the second stage of the tandem mass spectrometer and the resulting fragments are subsequently analyzed in the third stage of the tandem mass spectrometer, which may be a linear quadrupole, a TOF device, or otherwise as noted above. In broadband DIA (also known as “all mass/all ion MS/MS”), tandem mass spectra are acquired by fragmentation of all of the ions that enter the mass spectrometer at a given time. Alternatively, ranges of m/z of the precursor ions can be sequentially isolated, fragmented and then the fragments detected.
DDA, by contrast, seeks to confirm that one or more species is/are present in a given sample. Methods of DDA identify a fixed number of precursor ion species, and select and analyse those via tandem mass spectrometry. The determination of which precursor ion species are of interest in DDA may be based upon intensity ranking (for example, the top ten most abundant species as observed by peaks in a precursor mass spectrum, hereafter referred to as “MS1”), or by defining an “inclusion list” of precursor mass spectral peaks (for example by user selection), from which fragment spectra—hereafter referred to as “MS2”—are always acquired regardless of the intensity ranking of the peak in the precursor mass spectrum (MS1). Still otherwise, an “exclusion list” of peaks in MS1 can be defined, for example by a user, based e.g. on prior knowledge of the expected sample contents.
DIA avoids the decisions necessary in DDA, by simply dividing the mass range of interest (typically user defined) into segments and obtaining MS2 spectra for each segment (other than in the specific case of broadband DIA noted above). With DIA, the acquisition of an MS1 precursor spectrum becomes more or less optional, since the parameters of the precursor selection window themselves carry information about the range of possible precursor ions.
Early DIA techniques were disclosed in patent applications by Micromass UK Ltd and Water Technologies Corporation, in their so-called MSE arrangements. The DIA techniques resulted from application of known triple quadrupole methods to quadrupole-TOF arrangements.
In U.S. Pat. No. 6,717,130, a technique is disclosed in which MS1 and MS2 are alternatively acquired by repeatedly switching the energy of the fragmentation cell. The technique relies upon separation of the sample molecules through different elution times in a chromatography environment. At the end of an experimental run, precursor and fragment ions are recognized by comparing the mass spectra in the two different fragmentation modes. Fragment ions are matched to particular precursor ions on the basis of the closeness of fit of their elution times, so that precursor ions can then be identified.
U.S. Pat. No. 6,982,414 discloses a development to the DIA technique in the '130 patent described above. Here, the energy applied to the fragmentation cell is again repeatedly switched so as to obtain both MS1 and MS2. However here MS1 and MS2 are obtained from both a first and a second sample separately.
The mass spectra are then compared and further analysis is carried out where precursor ions in MS1 from each sample, or fragment ions in MS2 from each sample, are expressed differently.
Finally U.S. Pat. No. 7,800,055 again employs switching between energy levels in the fragmentation chamber so as to generate MS1 and MS2 in alternating manner. Comparison of the chromatographic peak shape of the precursor and fragment peaks is carried out to identify an association between precursor and fragment (product) ions.
An alternative approach to DIA, known as “SWATH”, has been proposed in various patents to DH Technologies Development Pte. Ltd.
In U.S. Pat. No. 8,809,770, a DIA data set is acquired such that the data may subsequently be analyzed for a target substance. This contrasts with the idea of setting a target and then acquiring data only for that purpose. The method employs LC-MS and uses wide windows of precursor ions (e.g. >10, >15, >20 amu) for MS2, allowing the whole precursor space to be covered.
Again the '770 patent stresses the importance of retaining the fidelity of the chromatographic peaks in the MS2 spectrum, by appropriate setting of the windows. An MS1 spectrum is indicated to be optional.
As an example, the '770 patent describes a method—akin to single reaction monitoring (SRM)—for evaluation of the MS2 data of a precursor mass window as a function of retention time, and for subsequent comparison with a reference spectrum library.
U.S. Pat. No. 8,809,772 employs isolation windows for the precursor ions, of variable width, the width being dependent upon the precursor mass. The method trades off analysis speed (for a wide window) and sensitivity/specificity (for a narrow window).
U.S. Pat. No. 9,343,276 addresses drawbacks with the methods disclosed in U.S. Pat. No. 8,809,770 by scoring extracted ion chromatogram (XIC) peak candidates based on various criteria, in a comparison between the XIC fragment peaks with the MS1 information, such as mass accuracy, charge state, isotopic state, known neutral losses and so forth.
A common aspect of the approach in the MSE and SWATH techniques described above is that they seek to optimize measurements for good MS2 time resolution. To obtain sufficient data points across the LC peak for good quantitation, either a relatively wide precursor isolation window (24 Da)—as suggested in the MSE patents discussed above—or a variable width precursor isolation window (the preferred approach for the SWATH patents discussed above) needs to be employed.
The consequence is that the traditional database search—in which sample fragment spectra are compared against fragment spectra of known species in a library, as is the case with DDA—may not be well suited for DIA data analysis. In the case of the SWATH technique, a large spectral library must first be created (for the same or similar sample types) for targeted extraction of MS2 chromatograms from convoluted spectra, which is an expensive and time consuming task.
In “MS1 Based Quantification Optimization on DIA Methods on a Quadrupole-Orbitrap Mass Spectrometer, Yue Xuan et al (available at https://tools.thermofisher.comrcontent/sfsiposters/PN-64738-MS1-Based-Ouantification-ASMS2016-PN64738-EN.pdf) a method of DIA analysis is disclosed in which a single MS1 scan is performed at a resolution of 120,000 across an m/z range of 350 to 1650 Th and a set of segmented MS2 scans across the whole mass range (350 to 1650) are performed at a resolution of 30,000 within a total duty cycle time of 2 seconds. The combined MS1 and MS2 duty cycle is repeated a number of times over the duration of an LC peak to provide a plurality of data points per peak for identification and quantification. The high resolution of the MS1 scan can distinguish the interferences from the analyte of interest.
Even then, for complex samples, the prior art DIA approaches require a high MS/MS scan speed and repetition rate, as well as relatively wide MS/MS windows, so that enough data points in the MS/MS domain are present for quantitation in the fragment domain, and for successful alignment between the MS1 and MS2 data streams. Such high speed throughput is not optimal for high resolution mass analysers.